Abstract

Insulin-like growth factor-II (IGF-II) is an embryonic growth promoter
and cell survival factor. IGF-II supply is normally limited by gene
expression because transcription occurs predominantly from the paternal
allele in mouse and man (maternal imprinting). Excess IGF-II has
detrimental systemic and local effects in vivo,
promoting somatic overgrowth and an increased frequency of tumors.
IGF2 mRNA is overexpressed in colorectal and many other
human cancers. In this paper, we show that altered IGF-II supply
modifies intestinal tumor growth. Mice genetically altered in the
IGF-II system were combined in crosses with
ApcMin/+, a murine model of human familial
adenomatous polyposis. Depending on genetic background,
ApcMin/+ acquires multiple small intestinal
adenoma before becoming moribund with anemia. Mice that express excess
IGF-II delivered using a bovine keratin 10 promoter
(k10Igf2/+) develop a disproportionate overgrowth of
colon, uterus, and skin. Combination with
ApcMin/+ leads to a 10-fold increase in
the number and the diameter of colon adenoma
(P < 0.0001) compared to
ApcMin/+ littermate controls (postnatal day
80), an increased susceptibility to rectal prolapse (41%), and a
histological progression to carcinoma. Mice with reduced IGF-II supply,
secondary to the disruption of the paternal Igf2 allele
(Igf2+m/−p), are 60% the weight of
wild-type littermates. Combination with
ApcMin/+ leads to a 3-fold reduction in
small intestinal adenoma number (P < 0.0001) compared to ApcMin/+ littermate
controls (postnatal day 150), and a significant decrease in adenoma
diameter (P < 0.001). With in
situ hybridization, we show that Igf2 was
expressed in all adenoma irrespective of IGF-II supply. This suggests
that there is an increased maternal allele expression of
Igf2 (loss of imprinting) in adenoma which form, despite
paternal Igf2 allele disruption. We conclude that IGF-II
supply is a modifier of intestinal adenoma growth, and we provide
genetic evidence for its functional role in colorectal cancer
progression.

INTRODUCTION

Mouse models of human cancer provide in vivo systems
for the identification of polygenic modifiers that may account for the
variation in penetrance, rate of tumor progression, and clinical
behavior of tumors. For example, our understanding of the genetics of
highly penetrant genes such as APC has been complemented by
the investigation of murine models, e.g.,
ApcMin/+, a close genotypic and phenotypic
model of human familial adenomatous polyposis. In familial adenomatous
polyposis, multiple colonic adenomas are frequent and commonly progress
to invasive carcinomas if not treated. The adenomas that develop in the
ApcMin/+ mouse can also progress to
invasive carcinoma, but the model differs from the human syndrome
because multiple adenomas tend to be concentrated in the small
intestine, which usually leads to anemia and intestinal obstruction
(1)
. The number of intestinal adenomas is dependent on the
inbred strain, which has been exploited in mapping a major modifier,
Mom1 (Pla2g2a) (2, 3)
. Although the
relevance of Pla2g2a to human colonic carcinoma progression
is still under investigation (4)
, its discovery enforces
the notion that the identification of genetic modifiers will define the
key interactions between biochemical systems that determine tumor
progression in vivo. We have taken a complementary approach,
which has been to use mice with a genetic alteration of a candidate
modifier known to be frequently disrupted in human cancer. In this
paper, we show that genetic manipulation of
IGF3
-II supply modifies the growth of intestinal adenoma in
ApcMin/+.

IGF-II is a paternally expressed, maternally imprinted, embryonic
growth factor that is a potent modifier of growth in vivo(5)
. In mice, after disruption of the paternal
Igf2 allele, total body weight is reduced by 40% at birth
(6)
. Systemic IGF-II levels fall after birth, and
postnatal growth is then regulated by the related ligand, IGF-I
(5)
. Increased systemic availability of IGF-II, either due
to biallelic expression, increased delivery from a transgene, or
disruption of the IGF-II/M6P receptor, results in overgrowth phenotypes
in mice (7, 8, 9)
. Similar effects occur in human overgrowth
syndromes such as Beckwith-Wiedemann, where the biallelic expression of
IGF2 results from either LOI or unipaternal disomy
(10)
. Increased expression of IGF-II in transgenic mice
not only increases tumor frequency in organs that express the
transgene, but also at distant sites, suggesting that both local and
systemic supply can promote tumor progression (11, 12)
.

IGF-II is well known for promoting a cell number increase in
vitro. The mechanism may predominantly relate to cell survival
rather than cell division (13, 14)
. IGF-II and the related
ligand, IGF-I, exert cell survival and growth effects via
heterotetrameric IGF-I and insulin receptors, which mediate signal
transduction through the PI3 kinase/Akt pathway also modified by the
recently identified phosphatase and tensin homologue deleted on
chromosome ten (PTEN) tumor suppressor (15)
. Serial
analysis of gene expression has identified IGF-II as the most abundant
mRNA overexpressed in human colorectal cell lines and tumors compared
to normal tissue (16)
. IGF-II is also overexpressed in
Wilms tumor, rhabdomyosarcoma, neuroblastoma, germ cell tumors,
adrenocortical carcinoma, breast, and hepatocellular carcinoma
(reviewed in Ref. 17
). Furthermore, increased maternal
allele expression (allele ratio of <3:1 taken as LOI) has also been
detected at surprisingly high frequency in normal human leukocytes and
colonic mucosa (12%), particularly in cases with microsatellite
instability in associated tumors (91%; Ref. 18
). This
significant finding suggests that increased local IGF-II supply may
predispose to the development of early onset colorectal cancer before
the appearance of either an adenoma or tumor, especially in individuals
with defects in DNA mismatch repair (19)
.

We examined the effect of IGF-II supply in crosses between mice with
genetically altered IGF-II expression and the
ApcMin/+ model of colorectal cancer.
Increased IGF-II delivery to the alimentary tract was achieved using a
bovine keratin 10 promoter-driven transgene (K10Igf2), which
results in the phenotype of colon, skin, and uterine overgrowth
(9)
. Although the predominant growth effect is in tissues
that express the transgene, there are also subtle metabolic effects due
to increased circulating IGF-II (20)
. Decreased IGF-II
supply in the alimentary tract was achieved using mice with a
disruption of the paternal allele of Igf2
(Igf2+m/−p) (6)
. Except for
the leptomeninges of the brain, Igf2 mRNA expression from
the maternal allele is normally undetectable in these animals.
Therefore, the source of any locally delivered IGF-II to the alimentary
tract should be derived from increased maternal allele expression.

MATERIALS AND METHODS

Mice.

ApcMin/+ (C57Bl/6J) were obtained from the
Imperial Cancer Research Fund. SPF colony (gift from W. Bodmer,
mice established in 1992 from A. Moser and W. Dove, McArdle laboratory,
Madison, Wisconsin; Ref. 21
) and bred in the host
department. Male ApcMin/+ mice were
backcrossed to inbred female C57Bl/6J (from Harlan, Bicester, Oxon,
United Kingdom) under non-SPF conditions for two generations before
commencing experimental crosses. Entamoeba muris was the
only intestinal parasite detected in both imported
ApcMin/+ and C57BL/6J stock. SPF conditions
do not significantly alter adenoma number (22)
. Animals
were housed with littermates under a 14 h light/10 h dark cycle
and were fed normal chow (3% total fat, Special Dietary Services,
Witham, Essex, United Kingdom) and tap water ad libitum.

Mice with increased IGF-II supply (K10Igf2) were at >10th
generation inbred onto 129/SvJ (9)
. Mice with disruption
of the paternal allele of Igf2 (−p) but an intact maternal
allele (+m, denoted Igf2+m/−p) were a gift
from A. Efstratiadis and were also at >10th generation inbred onto the
same 129/SvJ (6)
. DNA was extracted from tails (day 7) and
liver (at the time of dissection). After incubation (12 h; 55°C) with
0.5 μg/ml proteinase K in lysis buffer [50 mm
Tris (pH 8.0), 100 mm EDTA, 100
mm NaCl, 1% SDS] and RNase A (1 h; 37°C), DNA
was extracted with phenol/chloroform, precipitated with ethanol, and
resuspended in TE buffer [10 mm Tris (pH 7.4), 1
mm EDTA]. Animals were genotyped for the
presence of ApcMin/+, K10Igf2/+,
and Igf2+m/−p by established PCR protocols
(2, 23)
. All breeding used male 129/JSv IGF-II mutant mice
and female C57Bl/6J ApcMin/+. Female
K10Igf2/+ are poor mothers because they develop an
imperforate uterus, and the disrupted allele in
Igf2+m/−p is paternally inherited. All
litters were cross-fostered to F1 mothers before postnatal day 7
(C57Bl/6J,CBA/Ha or C57Bl/6J,129/SvJ). All animal procedures were
approved by the Home Office of the United Kingdom government,
departmental ethics committee and were carried out in accordance with
the United Kingdom Coordinating Committee on Cancer Research guidance
for the welfare of animals in experimental neoplasia (second edition;
Ref. 23
).

Adenoma Scoring and Collection.

Depending on age, ApcMin/+ become moribund
as a result of chronic anemia and intestinal obstruction. After daily
monitoring of initial litters for signs of anemia and distress, the
dates of dissection of experimental crosses were refined so that
animals did not suffer unduly. Small intestinal adenoma and colonic
adenoma were therefore scored for number and diameter either at
postnatal day 80 or 150 for the cross between
ApcMin/+ × K10Igf2/+
and ApcMin/+ × Igf2+m/−p, respectively. The stomach,
small intestine, and colon were dissected free of mesentery and opened
along the longitudinal axis using a jig and blade designed by us to aid
rapid processing. Intestinal contents were cleared with PBS, and the
small intestine was divided into three equal-length segments and laid
open with the colon on the absorptive side of Benchkote (Whatman).
Intestines were fixed in 4% (4 g/100 ml) paraformaldehyde in PBS (24
h) followed by 70% ethanol (v/v). Using a dissecting microscope
(×10–30) and calipers, adenoma number and diameter were obtained for
the entire length of the small intestine and colon. Adenoma analysis
was performed without knowledge of genotype by one person (A. B.
H) and confirmed independently by another (J. A. H.).
Material for cryosections was either placed face down relative to the
cutting surface for the small intestine or rolled (for the colon),
immediately embedded in TissueTek (Sakura Fintek, Zoeterwoude,
the Netherlands), and stored at −40°C. Small intestine surface area
was calculated by summation of the multiples of length of each fixed
segment by width, which was measured at the midpoint of each segment.
Colon surface area was calculated by multiplying the length of the
fixed material from the anorectal junction to the point of insertion of
the small intestine, omitting the appendix, by the width at the
midpoint. Statistical analysis is described in figure and table
legends. Calculations were performed using the Minitab 10Xtra (Minitab
Inc.).

Histopathology.

Distal colon samples and small intestinal segments were
paraffin-embedded and sectioned (5 μm), and every fifth section was
stained with H&E. Stained sections were viewed without knowledge of
genotypes (A. B. H) and checked by an independent histopathologist
(D. Rowlands, Department of Histopathology, University of Birmingham,
United Kingdom). Sections for immunohistochemistry were cleared with
xylene and rehydrated in an ethanol series to PBS, and endogenous
peroxidases were quenched with 3%
H2O2 in PBS (v/v; 15 min).
Sections were incubated at 4°C with primary antibodies in PAT
(PBS/0.1% BSA/0.1% Tween 20) for 16 h. The following antibodies
were used: antihuman APC (C-20) rabbit polyclonal to the COOH-terminus
of human APC (amino acid 2824–2843), antihuman APC (N-15) rabbit
polyclonal to the amino terminus of human APC (amino acid 2–16), and
IGF-IRβ (sc-713) antihuman rabbit polyclonal to a
carboxy-terminal peptide, all from Santa-Cruz Biotechnology Inc.
(Santa Cruz, CA). Proliferation was assessed with the HsMCM2/BM28
rabbit polyclonal antibody to human MCM2, a cell cycle protein marker
specific for G1-S-phase cells, a gift from I.
Todorov (24, 25)
. Sections were blocked with 2% (v/v)
horse serum in PAT (30 min), incubated with biotinylated antirabbit
antibody from horse (Vector Laboratories Inc., Burlinghame, CA), and
visualized with a peroxidase ABC Vector Elite kit with
3,3′-diaminobenzidine substrate. Sections were dehydrated,
counterstained with methyl-green, washed in acetone plus 1% (v/v)
acetic acid, and mounted.

RESULTS

Minor Modifier of ApcMin/+ in 129/SvJ.

We first established the total adenoma count in litters from
crosses between C57Bl/6J ApcMin/+ and
inbred stock 129/SvJ mice (coisogenic). Mean counts revealed a 2-fold
reduction in adenoma number at 100 days (14 adenoma;
n = 5) compared to C57Bl/6J
ApcMin/+ controls (31 adenomal;
n = 7), which is consistent with the presence
of a suspected semidominant modifier in the 129 strain
(27)
.

The bovine keratin 10 promoter used to deliver Igf2 mRNA
(K10Igf2/+) was previously found to target transgene
expression to the suprabasal layers of the skin, alimentary canal, and
uterus (9, 28)
. To our knowledge, this is the only
transgene available that increases IGF-II supply in the colon, although
a similar phenotype has recently been described for an actin-IGF-I
transgene (29)
. Overgrowth of the colon can result in
rectal prolapse in K10Igf2/+ mice that are >6 months old,
yet no intestinal epithelial tumors have been observed, even in the
highest expressing line used (“Blast” line). However, small raised
pale polyps in the distal colon can be seen in these animals (average
of 3.5 polyps, >2 mm in diameter/mouse colon at postnatal day 80;
n = 18). Histological analysis revealed
mucosal collections of lymphocytes (not shown). Therefore, all colon
polyps were checked by H&E-stained paraffin-embedded sections and
cryosections, and only nonlymphoid adenoma counts were reported.
Although K10Igf2 transgene expression has not been resolved
with respect to the four separate cell types of the crypt (see in
situ hybridization), there is evidence for both smooth muscle
thickening and increased crypt depth in the colon, indicating
overgrowth in both compartments (30)
. Apart from systemic
delivery from the blood stream, a further source of IGF-II may be
release from the stomach into the lumen and distal delivery to the
small intestine and colon. Both IGF-I and IGF-II may increase mucosal
cell growth after intraluminal supply (31)
.

Mice that develop intestinal adenoma and increased IGF-II supply
in the colon (ApcMin/+,K10Igf2/+)
lose weight (Fig. 1A)⇓
, rapidly become anemic, and commonly develop rectal
prolapse by 80 days (41%, 7/17 compared to K10Igf2/+ alone
6%, 1/18). To counter the possibility that altered adenoma growth was
simply a reflection of an alteration of small intestine and colon
growth, we corrected for surface area measured in fixed tissue. Adenoma
number and diameter were expressed either without correction (Fig. 2)⇓
or with correction for surface area (Table 1)⇓
. The number of adenoma increased in the colon (P < 0.0001; Fig. 2⇓
), even when correcting for increased colon
growth (P < 0.001; Table 1⇓
). The diameter of
the adenoma also increased disproportionately relative to the increased
colon growth (Table 2)⇓
. Examination of dissected colons revealed large distal adenomas (Fig. 3A)⇓
. Histopathological features within each adenoma showed a
spectrum of changes, with increased progression to carcinoma in
situ and invasion in a significant proportion (Fig. 3B⇓
;
Table 3⇓
). Only one polyp-like lesion was seen in the uterus of female
ApcMin/+,K10Igf2/+ (n = 7 animals); no mammary, skin, or stomach tumors were observed.

Effect of IGF-II supply on whole body weight (g). Male and
female mice were weighed at different times after birth until the time
of dissection, and results were pooled for each genotype (error
bars, ± SEM). A, increased IGF-II
supply using K10Igf2/+ transgene combined with
ApcMin/+. B, decreased IGF-II
supply using Igf2+m/−p combined with
ApcMin/+. n = number of mice per genotype.

Effect of IGF-II supply on number of adenoma. The
number of adenoma in the small intestine and colon and the combined
total adenoma count per mouse were pooled for each genotype after
dissection (day 80 and day 150 for
ApcMin/+ × K10Igf2/+ and
ApcMin/+ × Igf2+m/−p cross, respectively). Only
genotypes that developed adenoma are shown for clarity
(i.e., with ApcMin/+).
Results are expressed as box plots: box,
interquartile range; cross, median; verticle
line, 95% confidence interval. n = number of animals. NS, not significant. ∗,
P < 0.05; ∗∗,
P < 0.01; ∗∗∗,
P < 0.001; ∗∗∗∗,
P < 0.0001 (Mann-Whitney).

Intestines were dissected free, opened along the longitudinal axis,
cleaned in PBS, and fixed (4% paraformaldehyde). Surface area was
calculated by multiplying length by width at midpoint of fixed small
intestine and colon. Adenoma were visualized by using a dissecting
microscope (×10–30). Values are means±SD. Statistical comparison
between genotypes with normal and altered IGF-II supply utilized Mann
Whitney test.

Intestines were processed as for Table 1⇓
and adenoma visualized with a
dissecting microscope (×10–30) and size determined using fine
calipers. Genotypes with ApcMin/+ are shown only.
Values are means ± SD. Statistical comparison between
genotypes with normal and altered IGF-II supply was done with the Mann
Whitney test.

The majority of small intestinal and colonic adenoma >2 mm
showed reduced staining for Apc using a COOH-terminal monoclonal
antibody (25/31 adenoma from 13 animals), e.g., Fig. 4D⇓
. NH2-terminal APC antibody
stained all adenoma. There were no differences in Apc staining related
to IGF-II supply. Smaller adenoma tended to retain Apc staining in some
cells located within the adenoma rather than on its surface (Fig. 4A)⇓
. Proliferation visualized with an anti-MCM2 antibody
(BM-28) was confined to adenoma and basal crypts (Fig. 4, B and E)⇓
. Again, no differences in ratio of labeled:unlabeled
nuclei (proliferation index) in each high power field were detected
between adenoma of different genotypes (not shown). The distribution of
IGF1Rβ was concentrated in the smooth muscle layer, the upper luminal
surface of villi, and the upper zone of small intestine and colonic
crypts (Fig. 4C)⇓
. Large >2-mm colonic adenoma generally
appeared to have a ramifying network of staining confined to the
stromal cell compartment, rather than staining exclusively confined to
adenoma cells (Fig. 4F)⇓
. However, although a proportion of
adenoma had increased staining (18/31 adenoma from 13 animals), some
showed low level staining with anti-IGF1Rβ. No differences between
genotypes were detected.

Analysis of adenoma tissue. A-F,
immunohistochemistry of a small intestinal early adenoma
(A-C) and colon adenoma (D-F) from
ApcMin/+. Apc staining is commonly seen
within small adenoma but absent from large adenoma
(arrow; D), except cells forming edge of
adenoma (arrow head; D). Proliferation
occurs in adenoma as assessed by an antibody to human MCM2
(B and E; arrow). Crypt
cells also label (arrowhead; B). IGF-IR
(anti-IGF1Rβ) labeling occurs in crypts, villi, and smooth muscle
(arrowhead; F). Labeling also occurs
in cells at the edge of adenoma (arrowhead;
C) and as a ramifying network within adenoma
(arrow; F). G-I,in
situ hybridization with the Igf2 antisense probe
and sense probe (left lower insets). Adenoma from the
small intestine of ApcMin/+
(arrow; G);
ApcMin/+,Igf2+m/−p (two
adenoma; arrows; H); and
ApcMin/+,k10Igf2/+
(large arrow; I) all show a strong
signal. Colon crypts, and to a lesser extent, smooth muscle, show a
signal in ApcMin/+,k10Igf2/+
(small arrow; I). Bars,
100 μm.

Reduced IGF-II Supply Reduces Number and Diameter of Small
Intestine Adenoma.

Mice with reduced IGF-II supply
(Igf2+m/−p) were 60% of the weight of
wild-type littermates throughout postnatal life, as previously
described (6,, 32
; Fig. 1⇓
). There was significant reduction
in adenoma number and diameter in
ApcMin/+, Igf2+m/−p
in the small intestine at 150 days, even allowing for the reduction in
small intestinal growth (P < 0.001; Fig. 2⇓
;
Tables 1⇓
and 2⇓
). The reduction in adenoma size was most pronounced when
comparing adenoma with diameter >2 mm, suggesting that reduced IGF-II
supply limits early adenoma progression as well as total number (Table 2)⇓
. There were too few adenoma in the colon to detect a similar trend.
Histological comparison of small intestinal adenoma from
ApcMin/+,Igf2+m/−p
with
ApcMin/+,Igf2+m/+p
revealed similar histological features, with no differences independent
of adenoma size (not shown).

Igf2 Is Expressed in ApcMin/+ Adenoma, and
Maternal Igf2 Allele Is Expressed in
ApcMin/+,Igf2+m/−p.

Igf2 in situ hybridization of adult wild-type C57Bl/6J
villi, crypts, and smooth muscle layers of small intestine and colon
showed only background signal. In situ hybridization in
K10Igf2/+ (Blast) revealed Igf2 expression in the
upper two thirds of the crypts of the stomach and colon and associated
low level signal in smooth muscle layers (Fig. 4I)⇓
.4In situ hybridization in adenoma revealed an increased
signal in
ApcMin/+,Igf2+m/+p
(17/21 small intestinal adenoma from six animals),
ApcMin/+, K10Igf2/+ (10/12 colon
adenoma from six animals), and ApcMin/+,
Igf2+m/−p (9/13 small intestinal adenoma from
four animals; Fig. 4⇓
, G-I). Signal intensity appeared
similar irrespective of genotype in parallel processed slides. We
presume that mRNA degradation accounts for the failure to detect signal
in all adenoma because frozen section samples kept for longer than 3
months (at −20°C) frequently showed background signal.

DISCUSSION

Our results demonstrate that intestinal adenoma in
ApcMin/+ express IGF-II mRNA and IGF1Rβ.
Genetic manipulation of IGF-II supply significantly modified adenoma
growth in ApcMin/+. Increased IGF-II
supply led to a disproportionate increase in adenoma number, suggesting
either an increased rate of adenoma initiation or an increased rate of
early adenoma progression soon after initiating mutation. The effect
was greater in the colon rather than the small intestine, reflecting
the distribution of transgene expression used to deliver extra IGF-II
(9)
. K10Igf2/+ transgene expression is
relatively low in the small intestine compared to the colon, and small
intestinal adenoma, number, and size were not disproportionately
increased in mice with the combined genotype
(ApcMin/+,K10Igf2/+). This
observation makes it unlikely that systemic IGF-II levels significantly
altered adenoma growth. In
ApcMin/+,K10Igf2/+ mice, rectal
prolapse was prominent. We note that rectal prolapse has also been
observed in mice that also develop colorectal cancer due to a
homozygous disruption of Smad3 (33)
and in clinical
cases where rectal prolapse may lead to a 4-fold increased risk of
colorectal carcinoma (34)
.

Decreased IGF-II supply limited the number of adenoma in the
small intestine, again suggesting either a reduced rate of adenoma
initiation or a decrease in early adenoma progression. The disruption
of the Igf2 paternal allele had a clear effect on the number
of large adenoma, suggesting that IGF-II influences the growth of
established adenoma. The fact that adenoma appeared in the absence of
paternal Igf2 expression may be explained by either the
selection for the autocrine expression of Igf2 from the
maternal allele or by the expression of an alternative growth factor.
It is improbable that adenoma growth will depend on a single growth
factor such as IGF-II. However, results from Igf2 in situ
hybridization show adenoma-specific Igf2 expression and support
selection for increased maternal allele expression. Similar findings
have been described by Christofori et al.(35, 36)
in a pancreatic tumor model using SV40 T-antigen expression
from a rat insulin promoter (RIP-Tag) and in both TGFα and SV40
T-antigen-induced hepatocellular carcinoma models (37, 38)
. However, although SV40 T-antigen-induced hepatocellular
carcinoma showed reduced tumor size in combination with
Igf2+m/−p, increased maternal allele
expression was rarely found. Increased IGF-II supply in tumors with
intact Igf2 alleles appeared to be due to selection for
paternal allele disomy and maternal-specific LOH (38)
.
Studies of intestinal adenoma growth in mice with homozygous disruption
of Igf2 are in progress
(ApcMin/+,Igf2−m/−p).

The effects of increased and decreased IGF-II supply support our
view that IGF-II supply is a modifier of adenoma number and
progression. How IGF-II alters adenoma number is not known, but the
mechanisms could include either enhanced survival of cells that have
lost the normal Apc allele
(ApcMin/−, LOH) or via modification of an
increased crypt fission rate detected in the developing intestine of
ApcMin/+(39)
. The mitogenic
and apoptotic functions of C-MYC, transcriptionally
up-regulated as a result of APC dysfunction
(40)
, often require addition of survival factors, such as
IGF-II, particularly in c-myc-induced, p53-dependent, cell death
transduced by p19ARF(14, 41)
.
Mechanisms of how IGF-II acts as a survival factor include
phosphorylation and inactivation of Bad, which normally antagonize
Bcl-2 blockage of cytochrome c release (42)
.
IGF-IR-mediated cell survival functions may also be influenced by
systemic levels of the growth hormone-controlled ligand, IGF-I, because
an increased frequency of colonic carcinoma can occur in acromegalic
patients with excess IGF-I (43)
.

Patients with microsatellite instability in colorectal tumors
develop LOI of IGF2, which may promote growth of colon
tumors. However, it is not known whether LOI in this circumstance has
significant functional consequences in terms of increasing the
probability of developing early onset colorectal cancer. However, our
experiments highlight the potential importance of this observation and
of increased local IGF-II expression due to LOI in normal human colonic
tissue (18)
. It is not known whether increased IGF-II
supply in normal colon mucosa predisposes to colonic adenoma without
mutation of APC or whether IGF-II supply contributes to the development
of polyclonal adenoma via a paracrine/community effect
(44)
. We found no obvious increase in the proportion of
adenoma with normal Apc staining in mice with increased IGF-II supply.

Excess IGF-II expression is not the only perturbation of growth
factor pathways in colorectal cancer. Frequent mutations can occur in
growth factor receptor genes in human tumor-associated mismatch repair
defects, e.g., the TGFβ type II receptor (45)
and the IGF-II/M6P receptor (46)
. In addition to mutation
of the type II TGFβ receptor, Smad3 and Smad4 transducers of the
TGFβ pathway are also mutated in human tumors and result in
increased malignant progression of intestinal tumors after disruption
of murine genes (33, 47)
.

Igf2 expression and subsequent autocrine/paracrine
growth effects must offer adenoma cells a selective advantage. Our data
provide experimental support for mathematical models concerning natural
selection of expanding tumor cell clones expressing autocrine cell
survival factors (48)
. It is clear that IGF-II supply is
tightly regulated in normal tissue, with expression predominantly from
one allele during embryonic growth in both the human and mouse
(6, 49)
. The addition of a single-expressed
Igf2 allele results in overgrowth, and reduced IGF-II supply
results in reduced embryonic growth (−40%). We and others have shown
that IGF-II is an important regulator of murine tumor growth, both in
early adenoma and in the progression to carcinoma
(35, 36, 37, 38)
. However, this is the first demonstration of the
influence of IGF-II supply on intestinal tumor growth in a murine model
that closely mimics a human colorectal cancer syndrome and is
independent of the SV40 T antigen. We conclude that IGF-II supply is a
potent modifier of intestinal tumor growth and that IGF-II may
subsequently prove to be an important target for human colorectal
cancer therapy.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by a grant from The Cancer
Research Campaign United Kingdom 2390/0101 (to A. B. H.).
A. B. H. is a Cancer Research Campaign Senior Clinical
Research Fellow.

↵2 To whom requests for reprints should be
addressed, at the Department of Zoology, University of Oxford, South
Parks Road, Oxford OX1 3PS, United Kingdom. Phone: 44-1865-271227; Fax:
44-1865-271228; E-mail: bass.hassan{at}zoo.ox.ac.uk